Fred Hutchinson Cancer Center

University of Washington

The United States of America As Represented by the Department of Health

A Dusty Miller

David W Russell

Ian E Alexander

Lynn Bonham

Maribeth V Eiden

Ronald H Reeder

Carolyn A Wilson

Theodore D Palmer

Greg Wolgamot

Theo D Palmer

Mohammed A Adam

Jose V Garcia-Martinez

Brian M McStay

William Reginald Alfred Osborne

rDNA promoter constructs useful in plasmids and vectors directing transcription of RNAs in a Pol I-specific manner constructed of four elements in a serial array: a first nucleotide sequence, capable of hybridizing under stringent conditions to an rDNA promoter element; a second nucleotide sequence, capable of hybridizing under stringent conditions to an internal ribosome entry signal (IRES); a third nucleotide sequence containing a coding region interest; a fourth nucleotide sequence containing a polyadenylation (polyA) signal sequence; a fifth rDNA enhancer element may be positioned upstream of the serial array. Also recombinant permissive cells and genetically engineered stable cell lines that contain the subject constructs.

Polymerase I promoter plasmid and vector constructs

Retroviral vectors for producing coordinately expressed polycistronic mRNA in transfected host cells. A representative retroviral construct capable of forming a proviral genome in a host cell contains a first nucleotide coding sequence, a second nucleotide coding sequence, and a third nucleotide sequence capable of hybridizing under stringent conditions to a 5&prime; nontranslated region (NTR) of a picornavirus RNA or its complementary RNA strand. The first, second, and third nucleotide sequences are operably linked such that transcription of the proviral genome gives rise to a messenger RNA molecule containing transcripts of the first, second, and third nucleotide sequences. The transcript of the third nucleotide sequence in the messenger RNA molecule contains a nucleic acid capable of forming a regulatory stem-loop nucleic acid structure followed by at least one operable AUG start codon. The regulatory stem-loop nucleic acid structure is capable of operably binding a translation initiation complex in a host cell such that the transcripts of the first and second nucleotide sequences in the messenger RNA molecule are coordinately expressed in the host cell.

Cap-independent multicistronic retroviral vectors

Retroviral packaging cells produce replication-defective retroviral particles capable of binding to Mus dunni endogenous virus retroviral receptors on target cells and are useful in gene transfer and gene therapy. The packaging cell employs a vector encoding a M. dunni retroviral Env protein and produces the retroviral particles at high titer.

Mus dunni endogenous retroviral packaging cell lines

Method for increasing transduction of cells by adeno-associated virus

Retroviral packaging cells produce replication-defective retroviral vector particles capable of binding to Glvr-1 or Ram-1 retroviral receptors on target cells and are useful in gene therapy. The packaging cell employs a vector encoding a 10A1 retroviral env protein and produces the retroviral particles at high titer.

10A1 Retroviral packaging cells and uses thereof

The invention includes methods for increasing the efficiency of transduction of cells, including non-dividing cells, by recombinant AAV vectors. The methods utilize agents that alter certain aspects of DNA metabolism, more specifically, that affect DNA synthesis and/or affect repair, that impact on maintenance of chromosomal integrity, and/or that cause damage to the cellular DNA. Agents and vectors can now also be preselected and screened for transducing ability and/or transducing agents for their effect on DNA metabolism. These agents include tritiated nucleotides such as thymidine, gamma irradiation, UV irradiation, cis-platinum, etoposide, hydroxyurea and aphidicolin.

Retrovirus packaging cell lines PG13 (ATCC No. CRL 10686) and PG13/LNc8 (ATCC No. CRL 10685), and retroviruses packaged by said cells.

Retrovirus packaging and producer cell lines based on gibbon ape

A process of mammalian gene therapy. Explanted fibroblasts are genetically modified by introducing a retroviral construct containing a nucleotide sequence encoding for a therapeutic substance. The genetically modified fibroblasts are implanted into a mammalian subject.

Retroviral gene transfer into diploid fibroblasts for gene therapy

DNA constructs consisting essentially of the promoter, gag, pol, and env sequences of a helper virus useful for making retrovirus packaging cell lines that do not yield helper virus and do not transfer the packaging function. Such DNA molecules are constructed by deleting from the genome of a replication-competent retrovirus all cis-acting elements except for the tRNA binding site. Specifically, deletion is made of the packaging signal, the site for initiation of second strand DNA synthesis, the site required for translation of reverse transcriptase during first strand DNA synthesis, and the provirus integration signal. DNA construct pPAM3 (ATCC No. 40234) is a representative embodiment. A cell line containing such an altered viral genome does not transmit this virus or transfer the packaging signal, but will transmit high titers of other viral RNAs containing the proper cis-acting elements, including retroviral vectors designed to carry foreign genes. Cell line PA317 (ATCC No. CLR 9078) is a representative embodiment.

Growing community of inventors

Seattle, WA, United States of America

A Dusty Miller